Protein modification by the attachment of ubiquitin to cellular proteins is a key mechanism in regulating many biological processes. Ubiquitin is covalently attached to target proteins via an isopeptide bond between its C-terminus and a lysine residue of the acceptor substrate. Additional ubiquitins can be conjugated to any of the primary amines of ubiquitin to form a polyubiquitin chain on the substrate. Assembly of a chain of =4 ubiquitins linked together via Lys48 of ubiquitin marks cellular proteins for degradation by the 26S proteasome. Ubiquitination of proteins is achieved through an enzymatic cascade involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitinligating (E3) enzymes. Ubiquitination occurs when an E3 binds to both substrate and an E2 thioesterified with ubiquitin (E2~Ub), bringing them in proximity so that the ubiquitin is transferred from E2 to substrate. Among the most intensively studied E3s are members of the cullin-RING ligase (CRL) superfamily, which are regulated by a reversible covalent modification of the cullin with the ubiquitin-like protein, Nedd8. Deconjugation of Nedd8 from cullins is catalyzed by Csn5, the novel metalloisopeptidase subunit of the COP9-Signalosome (CSN). The breadth of the CRL family, which contains up to 350 members, raises profound questions about how the Nedd8 conjugation and deconjugation cycle is coordinated to effect specific regulation of individual CRLs. Indeed, there must be some mechanism to control deconjugation of Nedd8 to avoid simultaneous inactivation of all CRLs, which could be expected to have far-reaching and conflicting effects on cell regulation. Studies with the Nedd8-activating enzyme inhibitor MLN4924 reveal that the cycle of Nedd8 conjugation-deconjugation is extremely fast;within 5 minutes of administration of MLN4924, almost all of the Nedd8-modified cullins are deconjugated. Therefore, to truly understand the role of deneddylation it will be crucial to look at cells in the minutes following inhibition of CSN. To achieve this time resolution, genetic approaches are insufficient and it is essential to have a small molecule inhibitor. The specific aims proposed herein to achieve this goal are: (1) Assay development to complete transformation of the current fluorescence polarization 96-well baseline assay into a robust and reproducible 384 or 1536-well assay with values for S/B, CV, and Z'parameters suitable to support HTS and (2) Configuration of assays for HTS, including: (a) developing counter-screen and secondary assays and (b) validating the primary assay with the 1280-compound LOPAC collection.